A) Quantitative PCR and RT-PCR results for core July-G total archaeal 16S rRNA genes (black squares), MCG 16S rRNA genes (red stars), and MCG 16S rRNA (blue
squares). Shown are triplicate measurements for each sample. B) Ratio of average cDNA to average DNA of MCG per depth, with error bars showing standard error propagated from triplicate measurements of each sample. Boxed area denotes the middle of the SMTZ (see Figure 4-2d in Chapter 4 for methane and sulfate concentrations).
5.5 Discussion and Conclusions
The retrieval of MCG-derived 16S rRNA transcripts from station H in the White Oak River estuary indicates that MCG were likely active at all depths examined in July 2005 and 2008 and December 2006, with the exception of 3-6 cmbsf. The phylogenetic diversity of MCG does not appear to show patterns with sediment depth; sequences from different sediment layers are phylogenetically intertwined. Using an unfiltered 900 base pair
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615 MCG sequences from the 2007 Silva database release augmented by newer sequences from Genbank. (April 2009). The intragroup distance (29%) across all WOR MCG sequences nearly spans the distance of the larger database. This level of distance is larger than the intergroup distance (26%) of all methanogen lineages. This phylogenetic depth of the MCG group suggests that MCG might harbor similar or greater functional diversity.
The dominance of MCG in archaeal cDNA clone libraries is quantitatively substantiated by qPCR and qRT-PCR data showing that MCG is numerically dominant among the archaea (Table 5-2, Fig. 5-2a). Therefore, MCG appears to be active in White Oak River sediments, and its dominance among archaea implies that it might be a crucial
component of the microbial community here. The RNA content of MCG appears to be unreasonably low, at less than 10 copies of RNA per DNA copy (Fig. 5-2b). Several possible sources of error make the RNA and DNA comparison problematic. 1) RNA quantification requires an initial reverse transcription step, where secondary structure of the rRNA molecule may disrupt primer binding. 2) Much of the extracted RNA might be lost during processing due to its inherent instability relative to DNA. 3) Quantifications of DNA copy numbers might include DNA from inactive cells whose RNA is degraded, but whose DNA has yet to be degraded. Even if the absolute copy numbers of either RNA or DNA might be biased, the data allow inferences about changes within RNA and within DNA relative to depth and changes in RNA to DNA ratios with depth. 16S rRNA transcript copies of MCG drop below the SMTZ, while 16S rRNA gene copies of MCG remain constant. Therefore the ratio of RNA to DNA decreases below the SMTZ The shift to lower RNA content per DNA within the MCGs below the SMTZ may indicate that MCG are involved in organic matter
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recalcitrant. Or, perhaps this group relies on more oxidized electron acceptors that are in higher abundance in the sulfate reduction zone, such as sulfate or oxidized iron compounds.
The high diversity and abundance of archaea from the MCG suggest that they are important components of the sedimentary microbial community in the White Oak River estuary. The cosmopolitan distribution of the MCG archaea in different sulfate reducing and methanogenic sediments might suggest that these archaea show no clear geochemical habitat preferences within the anoxic sediment column. MCG have been found in anoxic terrestrial as well as anoxic marine environments including hot springs (Barns et al. 1996; Kanokratana et al. 2004), hydrothermal vent fluid (Nercessian et al. 2005; Huber et al. 2006), deep oceanic subsurface sediments (Parkes et al. 2005), deep terrestrial subsurface (Inagaki et al. 2003; Shimizu et al. 2006), continental shelf sediments (Vetriani et al. 1998), ancient marine sapropels (Coolen et al. 2002), petroleum contaminated soil (Kasai et al. 2005), termite guts (Friedrich et al. 2001), mud volcanoes (Heijs et al. 2007), methane hydrate-containing marine sediments (Inagaki et al. 2006), tropical hydrocarbon seeps (Wasmund et al. 2009), landfill leachate (Huang et al. 2003; Laloui-Carpentier et al. 2006), anaerobic wastewater reactors (Chouari et al. 2005; Collins et al. 2005; Roest et al. 2005), sulfidic springs (Elshahed et al. 2003), brackish lakes (Hershberger et al. 1996; Banning et al. 2005), and coastal salt marshes (Castro and Orgram 2004; Koch et al. 2006). Although the MCG seem to appear in clone libraries from nearly every type of sediment studied, there are some studies where large archaeal clone libraries do not contain MCG. These come from methane seeps (Knittel et al. 2005; Lloyd et al. 2006), and hydrothermal vents (Reysenbach et al. 2000; Huber et al. 2002), but many of these studies used the archaeal reverse primer A958r, which is biased against MCG (Teske and Sørensen 2008). However, Inagaki et al. 2003 found a preference of
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the MCG for volcanic ash layers and Coolen et al. found them primarily in sapropel layers, suggesting a preference for organic matter rich sediment layers. Consistent with this habitat preference, a heterotrophic metabolism is suggested by the carbon isotope ratios of lipids and whole cells extracted from MCG-rich deep oceanic subsurface sediments (Biddle et al. 2006). Our 16S rRNA transcription data suggest that MCG are found throughout the sulfate reducing and methanogenic parts of the sediment column the transcript to gene ratios are highest in the lower sulfate reduction zone and in the sulfate reduction zone. It is therefore likely that MCG are a ubiquitous, numerically dominant group of anaerobic heterotrophs that can access a wide variety of substrates (given their phylogenetic diversity and wide
distribution). This possibly large contribution to post-depositional carbon remineralization makes MCG a useful target for future culturing and ecophysiological research.